The genus Sebastes is a large group of saltwater fishes (>100 species currently recognized) that inhabit a diverse array of ecological niches in the marine environment (Love et al. 2002). Rockfish have been intensively studied from a genetics perspective (Burford and Bernardi 2008; Buonaccorsi et al. 2004; Gilbert-Horvath et al. 2006; and others) as they are part of many commercial and recreational fisheries and serve as useful models for studying marine systems. Here we describe a set of expressed sequence tag (EST)-microsatellites that can be used to explore ecological/evolutionary questions and supplement the conservation and management efforts for this group of fishes.

We used three independent EST datasets for microsatellite discovery. The first two sets of EST sequences were downloaded from GENBANK for S. rastrelliger and S. caurinus (accession numbers: GE796994-GE820661 and EW975926-EW987132). Given the close affinity of S. caurinus and S. rastrelliger (Hyde and Vetter 2007), we assembled ESTs from both species with CAP3 (Huang and Madan 1999) using default parameters. The third set of ESTs used for microsatellite discovery was developed from S. goodei ESTs(ovary and testes tissue—Aguilar et al. in prep). All S. goodei ESTs mined for microsatellites were previously checked for quality, trimmed and assembled with CAP3 (Aguilar et al. in prep). Microsatellite searches were preformed with TANDEM REPEATS FINDER v4.04 (Benson 1999) and primers were designed with PRIMER3 v0.4.0 (Rozen and Skaletsky 2000).

Microsatellites were amplified using a three-primer amplification which includes a fluorescently-labeled M13 primer (Schuelke 2000). Reactions were run in 15 μl volumes with the following conditions: 1X ABI buffer, 2.5 mM MgCl2, 0.4 mM dNTPs, 2.7 × 10−4 mg/ml BSA, 0.3 μM reverse primer, 0.3 μM fluorescently-labeled M13 sequence (5′-CACGACGTTGTAAAACGAC-3′) using Applied Biosystems Inc. (ABI) dye labels (FAM, VIC, NED, PET), 0.07 μM M13 5′-end labeled forward primer, 0.2 units of ABI Taq polymerase, and 5 μl of DNA (5–20 ng/μl). We used the following thermal profile: 95°C for 3 min, followed by 10 cycles of 94° for 30 s, 58°C for 45 s (‘touching down’ 1°C each cycle), 72°C for 45 s. This was followed by 35 cycles at 94°C for 30 s, 48°C for 30 s, and a 72°C for 45 s, and a final extension of 72°C for 7 min. Hundred and eleven novel primer pairs were tested on three rockfish species: S. goodei (N = 3), S. flavidus (N = 3), and S. melanops (N = 2). Products were run on an agarose gel for confirmation of amplification and those that displayed positive amplifications were run on an ABI3100 automated sequencer with LIZ-500 and genotyped using GENEMAPPER v4.0 (ABI). A total of 51 (out of 111) loci gave reliable and scorable products that we explored for within population variation (Table 1).

Table 1 Primers for 51 polymorphic EST-derived microsatellites
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Forty eight S. flavidus and 48 S. melanops samples from two California (USA) populations (north and south of Cape Mendocino respectively) were used to assess population level variation using the aforementioned PCR protocol. Products were typed on an ABI3100 as above. Departures from Hardy–Weinberg equilibrium, allele counts (k), and levels of heterozygosity (H exp , H obs ) were calculated using the program GENEPOP v4.0 (Rousset 2008). Loci that amplified and were scored successfully were annotated to the SWISSPROT database using the BLAST2GO software (Conesa et al. 2005). Data is only presented from loci that we were able to successfully amplify and genotype in S. flavidus and S. melanops. Primer information for loci that did not give successful amplifications, or were difficult to genotype, are available from the corresponding author.

After omitting fixed loci allele counts for S. flavidus and S. melanops ranges from 2 to 19 and 2 to 13, respectively. Observed heterozygosity ranged from 0.042 to 1.0 S. flavidus and 0.042 to 0.913 in S. melanops. Seven microsatellite loci did not conform to Hardy–Weinberg expectations (P ≤ 0.05) in S. flavidus, while five loci did not conform to Hardy–Weinberg expectations (P ≤ 0.05) in S. melanops (Table 2). Two primer sets appear to be amplifying duplicated loci (Sgoo690_1 and Sgoo9_832_1) due to the fact all individuals that successfully amplified were heterozygotes (Table 2).

Table 2 Sample size (N), observed number of alleles (k), observed (Ho) and expected (He) heterozygosity for two populations of S. flavidus and S. melanops
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Eleven loci were annotated to the SWISSPROT database. The remaining 40 loci did not show any significant matches. Two of the loci (Sgoo10_911_1 and Sgoo2027_1_ annotated to the same protein (Protein LBH) but were not in linkage disequilibrium (data not shown). These EST-linked microsatellites with annotations may serve useful in the development of a linkage or genomics maps of the Sebastes genome and further assist in the management and conservation in this economically important group. Additionally, gene associated markers provide opportunities for evolutionary analysis of locally adapted genes in multiple Sebastes populations. This is the first set of EST-linked microsatellites in Sebastes and shows that there is significant cross-species amplification among other species in the genus.